The cell cycle is usually divided into four phases: DNA synthesis (S phase) and mitosis (M phase) separated by gaps called G1 and G2. The cell cycle is coordinated by several Ser/Thr protein kinases that are activated in a regulated manner. It is this progressive activation and inactivation of a family of Cyclin-dependent kinases (Cdk's) that regulates the cell cycle. This enzyme engine is subject to careful control in order to ensure that each event (DNA replication, nuclear envelope breakdown, spindle formation, and chromosome segregation etc) is performed correctly and in proper sequence.
Each protein kinase consists of a catalytic cyclin-dependent kinase (Cdk) subunit and a regulatory cyclin subunit. Cdks are proteins that contain the catalytic subunit (.about.34 kDa) and are inactive as monomers. The best characterized Cdk's, in terms of their temporal activation and cognate cyclins, are: Cdc2, Cdk2, Cdk4, and Cdk6.
Cdk activation requires their specific association with the regulatory subunits called cyclins. In human cells different types of cyclins have been idenitfied and these include types A, B, C, D, and E. Cyclins may be further grouped into "Mitotic Cyclins," which include cyclins A and B and "G1 Cyclins" which include cyclins C, D, and E. The cyclins D, E, A, and B are required in G1, G1/S, S, and G2/M phases of the cell cycle, respectively.
Cdk activation requires both cyclin binding and phosphorylation by a Cdk-activating kinase (CAK) on Thr161 of the Cdk catalytic domain. Negative regulation of Cdk is carried out through phosphorylation of Thr14 and Tyr15. The phosphorylation and dephosphorylation at these residues is mediated by several enzymes including the Wee1 kinase and Cdc25 phosphatase.
Inactivation of the cyclin-dependent kinases (Cdks) is potentially a promising route to cancer therapy and has been the subject of immense interest for the last few years. These kinases belong to a family of enzymes involved in the the events that control the eukaryotic cell cycle.
Cdc2 and Cdk2 are two particularly well understood Cdks. The major partner for Cdc2 in the cell is cyclin B. Cyclin B levels peak at the G2/M transition and the protein is involved in the induction of mitosis. Cdk2 is active in late G1 and remains active until the end of G2. The levels of cyclin E peak around the G1/S transition during the cell cycle.
The dimunition of cyclin protein levels is an important aspect of the regulation of the cell cycle in normal cells. This reduction is thought to occur by targeted protein degradation. Indeed, most of the known cyclins contain one or the other of two classes of putative degradation motifs. These motifs are regions of the proteins containing a defined amino acid sequence. Mitotic cyclins, which include cyclins A, and B, contain a so-called "destruction box," whereas G1 cyclins, which include cyclins C, D and E, contain potential "PEST" motifs.
The PEST motif is a stretch of amino acids having a specific composition, and its presence in some proteins is known to cause the rapid degradation of eukaryotic cells. The mutation of these motifs in a cyclin would thus result in the abnormal persistance of high levels of cyclin, and this may contribute to tumorigenesis.
Protein inhibitors of Cdk/cyclin complexes mediating cell cycle progression have been discovered. The mammalian Cdk protein inhibitors fall into two categories based on sequence homology. One class is related to p16, and includes specific inhibitors of Cdk4 and Cdk6. The other class, related to p21, includes p21, p27 and p57.
The inhibitors related to p21 are known as the cyclin-dependent kinase inhibitory proteins (Kips). "Kips" used without a specific number identification usually refer to a family of proteins. Some proteins within that family may be given a more specific "name" that includes the letters kip or even cip, such as, p27-KIP1 or p21-CIP1. Capitalization may vary, e.g. kip, Kip, KIP or cip, Cip, CIP. Kips inhibit a wide variety of complexes including Cdk4/Cdk6-cyclin D, Cdk2-Cyclin A/Cyclin E, and cdc2/cyclin B. Human p21 (Cipl) encodes a protein of 164 amino acids and human p27 (Kip1) encodes a protein of 198 amino acids.
The Cdk2/cyclin E complex and associated cell cycle regulatory functions is an important research area and the search for inhibitors of this complex is an important research goal. If soluble, active complexes of Cdk2/cyclin E could be created, they would fill a critical need for a tool to create in vitro screens designed to detect inhibitors of the kinase activity of this complex and they might facilitate studies designed to characterize the complex and elucidate its properties.
The invention disclosed herein discloses various forms of cyclin E that create such soluble and active Cdk2/cyclin E complexs.